Organs and tissues of Rhynie chert plants

Abstract

The Early Devonian Rhynie chert and the nearby Windyfield chert contain the oldest in situ preserved terrestrial ecosystem. Two of the seven species of anatomically preserved land plants had naked axes, one an axis with a more or less regular pattern of short-longitudinal ribs, two species had spiny axes and one species had small leaf-like appendages. All plants mainly consist of parenchymatous tissues. In some species, conducting elements comprise uniformly thickened thick-walled cells resembling hydroids of larger bryophytes, whereas others have real tracheids with annular and/or spiral secondary wall thickenings. True phloem has never been demonstrated but in all species the thick-walled water-conducting cells are encircled by a zone of thin-walled cells without intercellular spaces. The cortex is differentiated into two or three zones and forms the major part of the axes; in one species the cells of the middle cortex are sclerified. Some species have a hypodermis. In all species the epidermis is covered by a well-developed cuticle. Sporangia are known from all species. Sporangia are spindle-shaped, lobed or kidney-shaped and attached terminally or laterally with a short stalk. Gametophytes of four species have been described. Gametophytes are unisexual, isomorphic but much smaller than the sporophytes.

This article is part of a discussion meeting issue ‘The Rhynie cherts: our earliest terrestrial ecosystem revisited’.

1. Introduction

In their landmark monograph on the Rhynie chert (1917–1921), Kidston & Lang [1–5] described five land plants with conducting tissues—some with reproductive organs, others without. Although Devonian plants were known since the mid-nineteenth century [6], the monumental work of Kidston & Lang was a real milestone, because it was the first time that plants showing such a great amount of anatomical detail were described from rocks as old as Early Devonian. The importance of the Rhynie chert was recognized immediately and can hardly be overestimated. The third and fully revised edition of D.H. Scott's authoritative ‘Studies in Fossil Botany’ [7] contained a whole new chapter on Rhynie chert plants, which were then still classified within the Psilophyta, even though the last two parts of Kidston and Lang's monograph still had to appear. Later, one of the forms described by Kidston & Lang was recognized as a new taxon [8].

Most of the Rhynie chert plants look quite simple and superficially similar at a first glance. All taxa have dichotomizing axes and are leafless, but the most advanced, Asteroxylon mackiei Kidston et Lang 1921, had veinless leaf-like enations. The aerial axes of all Rhynie chert plants primarily consist of parenchymatous cells; the conducting strand always constitutes only a relatively small proportion of the stem. Aglaophyton (Rhynia) majus (Kidston et Lang 1921) Edwards 1980, one of the most common constituents of the Rhynie flora, was described as a plant with dichotomizing axes and terminal spindle-shaped sporangia. Rhynia gwynne-vaughanii Kidston et Lang 1917 is similar but smaller and is characterized by being more profoundly branched and having true tracheids and so-called hemispherical projections. Hornea lignieri Kidston et Lang 1920, which was later transferred to Horneophyton [9], has bulbous rhizomes and branched sporangia with a central columella. Kidston & Lang [4] misinterpreted the fertile axes of a plant that was later described as Nothia aphylla [8] as belonging to Asteroxylon mackiei.

Although the first systematic description of Rhynie chert organisms appeared 100 years ago, the interest in the chert has never ceased and important new discoveries are still being made. In 1980, Remy & Remy [10,11] discovered an antheridia-bearing gametophyte. In later years, several other gametophytes were described from the Rhynie chert [12–15]. Fully developed gametophytes have a conducting strand, stomata and a cuticle. They were free-growing plants, their vegetative parts resembling the sporophytes, but they were much smaller. Of two species successive stages of the life cycle have been documented in great detail, which further underscores the uniqueness of the fossil flora from the Rhynie chert.

In later years, two additional taxa were described from the Rhynie chert and the nearby Windyfield chert, namely as Trichopherophyton teuchansii Lyon et Edwards 1991 and Ventarura lyonii Powell, Edwards et Trewin 2000 [16,17]. These latter two taxa are less well known and, therefore, this contribution will primarily focus on the species originally described by Kidston & Lang [1–3]. Although vegetative reproduction by means of rhizomes was recognized nearly a century ago [4], another way of vegetative reproduction was described recently [18]. Rhynie chert plants are often regarded as simple or primitive, but studies carried out in the last couple of decades show that they were far more complex than initially thought.

Rhynie chert plants are often preserved in situ. Apart from the plants described here, a rapidly growing number of microorganisms notably fungi, and various aquatic and terrestrial animals have been described from the Rhynie and nearby Windyfield cherts. The fact that Rhynie chert plants are prominently figured in nearly all textbooks on palaeobotany, systematic botany and Earth history demonstrates the great importance of this Konservat-Lagerstätte. A century after its discovery the Rhynie chert still continues to provide new information on early land plants. Therefore, this brief review represents the current state of knowledge, anticipating that more exciting discoveries will follow.

2. Organs and tissues

The most recent overview of the land plants from the Rhynie chert is given by Edwards [19], and deals with the sporophytes. Although the number of different organs and tissues is limited, and basic building plans look quite similar in most taxa, the Rhynie chert plants show a rather wide diversity in the combination of features that are described below in a number of sections. Each section deals with a specific organ or tissue. These characterizations are based on previously published studies supplemented by my own observations. In order to restrict the number of multiple references and unnecessary repetitions in the running text to a minimum, the primary literature sources for the individual taxa are listed at the end of each section.

Unfortunately, not all taxa and not all parts and tissues of the Rhynie chert plants are equally well preserved. The plants that grew in the most humid habitats are usually best preserved. The basal parts of others that grew on better-drained sandy substrates may be well preserved but the aerial axes are often not. Soft tissues like the cortex are usually (partially) decayed in plants of the latter category. Because the plants are studied from thin sections, sections in different directions, transverse and longitudinal, are needed for reconstructing a three-dimensional picture. Particularly, problematic are reconstructions of complex three-dimensional structures like branching patterns and the sporangia of Asteroxylon and Ventarura. This is only possible by making serial sections. Moreover, it is often difficult to evaluate certain aspects such as the cell pattern of the epidermis, because larger portions of the epidermis lying parallel to the cutting plane are rarely encountered. Owing to these limitations some of the following descriptions are shorter and less detailed than others. When given, cell sizes are usually approximations, because in many cases cutting planes are not ideal but oblique and post-mortem shrinkage may have occurred.

Thin sections are most appropriate for studying anatomical details of Rhynie chert plants. Slides thicker than the standard petrographic thin sections are preferred because they reveal at least some of the three-dimensional structure. Cell walls are often very thin and the organic matter has largely been replaced by silica. Attempts to study Rhynie chert plants by making cellulose acetate peels after etching the chert with hydrofluoric acid were not really successful. In peels only the most durable parts consisting of coalified residues, such as the thick-walled cells of the water-conducting strand, the cuticle, the epidermis and the sporangia are still visible. The best way to make thin sections is to start with a relatively thick section, usually over 200 µm thick, depending on the transparency of the chert. A slice of chert is mounted on a glass slide with thermoplastic synthetic resin and ground with Silicon carbide powder. The chert wafer can be released from the glass slide after heating, turned around and remounted in order to be ground down from the other side. If necessary this process can be repeated a few times until the amount of chert above and below the subject is minimized and the desired thickness is reached [20].

Even in thin sections cellular details may sometimes be difficult to observe. In cases when cell walls were very thin and hardly visible in transmitted light, the use of incident light and placing a piece of milk glass under the slide may reveal the finest details [21]. For our studies, attempts to apply other methods had only limited success. The use of fluorescence and confocal microscopy mostly failed because the silica matrix fluoresces stronger than the plant remains, which in many cases do not show any fluorescence at all, not even the cuticles, suggesting that only coaly material is present. Only very few of the several attempts to apply micro-CT scan were more or less successful.

A selection of material showing various tissues and organs is shown in figures 1–5. All illustrated slides are stored in the collection of the Forschungsstelle für Paläobotanik in Münster.

Figure 1.

(a,b,f) Rhynia gwynne-vaughanii: (a) cross-section of a young axis, (b) detail showing the different tissue types from the water-conducting strand (right) to the epidermis with the overlying cuticle (left), (f) water-conducting cells in longitudinal section; (c–e) Aglaophyton majus: (c) cross-section, (d) detail showing the different tissue types from the water-conducting strand (right) to the epidermis with the overlying cuticle (left), (f) water-conducting cells in longitudinal section showing wall thickenings; (g–h) Horneophyton lignieri: (g) bulbous rhizome and basal parts of upright axes, (h) water-conducting cells with annular to slightly helical wall thickenings in the basal part of the upright axis shown in (g). The dark ring to be seen in cross-sections (a–d) several cell layers below the surface consists of mycorrhizal fungi filling the intercellular spaces. Slide numbers: (a,b) P 5007; (c,d) P1870; (e) P 1876; (f) P 1588; (g,h) P 5012. Scale bars: (a,d) 500 µm, (b) 200 µm, (c) 1 mm, (e,h) 25 µm, (f) 50 µm; (g) 2 mm.

Figure 2.

(a–c,e) Aglaophyton majus, (a) strand of water-conducting cells surrounded by parenchymatic cells, (b) epidermis and hypodermis, (c) section through a stoma showing the substomatal chamber and cutinization of cell walls facing the stomatal channel and substomatal chamber, (e) stoma in the surface view; (d,f) Rhynia gwynne-vaughanii, (d) tracheids (dark) surrounded by parenchymatous tissue, (f) section through a stoma showing the substomatal chamber. Collection numbers: (a) Pb 5005; (b) Pb 1809; (c) Pb 1826; (d) Pb 5006; (e) Pb 1603; (f): Pb 1809. Scale bars: (a) 200 µm, (b) 100 µm, (c,f) 50 µm, (d) 25 µm, (e) 20 µm.

Figure 3.

(a) Rhynia gwynne-vaughanii hemispherical projection; (b–d) Nothia aphylla: (a) cross-section through a rhizome with rhizoids, (b) cross-section through an aerial axis, (c) detail of rhizoids; (e) Asteroxylon mackiei arrested apex of an aerial axis; (f,g) Horneophyton lignieri: (f) part of a nearly empty sporangium with central columella, (g) spore tetrad inside sporangium. Collection numbers: (a) Pb 2336; (b,d) Pb 2868; (c) Pb 2811; (e) Pb 4159; (f) Pb 2419; (g) Pb 2422. Scale bars: (a) 100 µm, (b,c) 500 µm, (d) 100 µm, (e) 250 µm, (f) 1 mm, (g) 20 µm.

Figure 4.

(a–g) Asteroxylon mackiei: (a) longitudinal section through a sterile axis with ‘leaves’, (b) cross-shaped xylem strand of an aerial axis, (c) tracheids with well-developed wall thickenings, (d) cuticle with stomata of a rhizome, (e) longitudinal section through a fertile axis with sporangia inserted between normal ‘leaves’, (f) cross-section through the tip of a fertile aerial axis—the dark structure in the lower right corner is a sporangium, (g) a sporangium still partly filled with spores. Collection numbers: (a) Pb 2365; (b) Pb 3404; (c) Pb 2367; (d) Pb 4001; (e) Pb 4113; (f) Pb 4123; (g) 4112. Scale bars: (a) 1 mm, (b,g) 500 μm, (c) 25 μm, (d) 250 μm, (e) 2.5 mm, (f) 1 mm.

Figure 5.

(a–i) Gametophytes of Rhynie chert plants: (a–d,f,g) Lyonophyton rhyniense, the gametophyte of Aglaophyton majus: (a) germinating spore, (b) young gametophyte with remnants of the spore, (c) mature antheridia-bearing gametophyte, (d) two antheridia, (f) mature antheridium releasing sperm cells, (g) longitudinal section through archegonium showing neck canal and egg chamber; (e) Kidstonophyton discoides, the gametophyte of Nothia aphylla; (h,i) Remyophyton delicatum, the gametophyte of Rhynia gwynne-vaughanii; (h) in situ stand of gametophytes, (i) gametophyte–sporophyte junction, the left axis is gametophytic as is evidenced by the presence of an archegonium (arrow), the sporophyte axis is characterized by the larger cell size. Collection numbers: (a) Pb 1251; (b) Pb 1693; (c) Pb 0021; (d) Pb 0023; (e) Pb 0013; (f) Pb 0011; (g) 3386; (h) Pb 3684; (i) 3679. Scale bars: (a,b) 40 μm, (c,e) 1 mm, (d) 200 μm, (f) 50 m, (g) 30 μm, (h) 2 mm, (i) 300 μm.

3. ‘Rooting’ structures and rhizomes

Rooting structures are essential parts of most higher land plants. However, in many cases, very little is known about the rooting structures of fossil plants, because roots are often found isolated and cannot be correlated with the aerial parts of the plant. None of the Rhynie chert plants has real roots. Even the most advanced species, Asteroxylon mackiei, which has repeatedly bifurcated root-like axes penetrating the substrate, lacks a true calyptra or root cap. Nevertheless, at least some different ‘rooting’ types can be distinguished in Rhynie chert plants.

The simplest are unicellular rhizoids of plants lying on the substrate that developed where the axes touched the surface of the substrate. Aglaophyton majus and Rhynia gwynne-vaughanii did not have underground parts. They were lying on the substrate, which could vary from a sinter surface to a sandy soil. When the upright aerial axes of Aglaophyton became too long and the vascular strand could no longer support upright growth, they bent down and touched the ground where they found support. Then they bent again and grew upward, resulting in the typical U-shaped growth. The regular distribution of the stomata all over the axes makes it unlikely that the rhizoid-bearing axes were primarily lying on the substrate as depicted in the original reconstruction [4]. Where axes touched the substrate, rhizoids developed near stomatal cells where cells were apparently more meristematic than the normal epidermal cells. Remy & Hass [22] stressed that axes touching the substrate are slightly different from upright aerial axes, e.g. in cell sizes, but they did not find any primary rhizome axis in the rich material at their disposal.

The second growth type comprises plants with (partly?) subterranean rhizomes. Subterranean rhizomes have the benefit that they can be used for storage and enable the plant to survive during less favourable periods. When conditions became more favourable new aerial axes developed from the underground rhizomes. Plants with subterranean rhizomes were primarily bound to habitats with a loose substrate such as sandy and clayey soils and peats. Nothia aphylla has repeatedly bifurcating rhizomes that are more or less pear-shaped in cross-section (figure 3b,d). Unicellular rhizoids developed from a median rhizoidal ridge on the ventral side of the rhizomes. Almost each ventral epidermal cell bears a rhizoid; the rhizoids depart radiating from the central ridge. The rhizoidal ridges are interrupted by narrow transverse incisions, about 100 µm wide. The rhizoidal ridges have a three to seven cell layers thick hypodermis consisting of parenchymatous cells without intercellular spaces. The rhizome has a central core of conducting tissue consisting of a central strand of thick-walled cells surrounded by a zone of thin-walled cells lacking intercellular spaces. These conducting tissues are partly encircled by a horseshoe-shaped cortex consisting of loosely packed parenchymatous cells with very prominent intercellular spaces. The rhizoidal ridges are connected with the oval central core by a so-called ‘connective’ primarily consisting of densely packed smaller parenchyma cells with some thick-walled cells that either occur isolated, interspersed between the other cells, or that are partly connected to each other, forming an irregular ventrally directed strand. Some blocks and sections cut perpendicularly to the stratification show up to four successive horizons with rhizomes in a sandy soil, all showing very good cell preservation of the parenchymatous tissues, but the arising aerial axes are partly decayed. This indicates that the rhizomes were long-living, still alive shortly before silicification and fully covered by soil.

Horneophyton lignieri has a basal bulbous corm lacking conducting tissues (figure 1g). The conducting strand starts shortly above the transition of the corm to the aerial axis. The ventral side of the corm bears numerous, radially diverging unicellular rhizoids. Essentially parenchymatous axes of Ventarura lyonii with rhizoids all around are suggested to have been subterranean.

The third type is only known from Asteroxylon mackiei that had leafy rhizome axes lying on the substrate that gave off subterranean geotropic axes that bifurcated a few times and penetrated the substrate. The root-like axes have two whorls of small, downward-projected scales inserted just below their departure; the rest of the root-like axes is smooth. Details on the vascularization of the rhizome and rooting axes are given below (6a(i)).

For detailed descriptions of and discussions on ‘rooting’ structures and subterranean axes see:

Aglaophyton majus: [22,23]; Horneophyton lignieri: [2]; Nothia aphylla: [24–26]; Asteroxylon mackiei: [3]; Ventarura lyonii: [16].

4. Branching modes in Rhynie chert plants

The dichotomy is the basic branching mode of all Rhynie chert plants. The aerial axes of Aglaophyton majus, Horneophyton lignieri, Nothia aphylla, Ventarura lyonii and probably also Trichopherophyton teuchansii are isotomously branched. However, arrested apices occur in Aglaophyton. They are rare on the upright aerial axes but may be common in the basal portions of the aerial axes, particularly in the U-shaped parts where they rested on the substrate. The axes produced wart-like bulges of smaller radially arranged cells. Arrested apices ultimately formed adventitious axes or bulbil-like organs. The adventitious axes and the parent axis are often not connected by conducting tissue. The bulbil-like organs grew out to bowl- or disc-shaped structures from which aerial axes developed. The dark layer that may be present between the parent axes and the bulbil-like organs may indicate that they were abscised and served for vegetative reproduction.

Rhynia gwynne-vaughanii was originally reconstructed as a simple, sparsely dichotomously branched plant with occasional shorter adventitious branches [2]. Edwards [27] demonstrated that the branching pattern is more complex and that Rhynia was far more branched than suggested by Kidston & Lang [4]. Dichotomies are comparatively rare and adventitious branches are prevalent. Adventitious branches occur over the entire axis, including overtopping axes inserted just below the sporangia.

Asteroxylon mackiei is reconstructed as a plant with monopodial upright leafy aerial axes arising from a rhizome. The lateral axes branch dichotomously. Root-like axes depart from the rhizome [3,28]. Arrested apices are randomly distributed on the rhizome and the aerial axes and appear as small shoots with young leaves; these leaves are, depending on the stage of development, still partly coiled (figure 3e).

In all Rhynie chert plants the outline of the axis becomes slightly elliptical just below the bifurcation. The outline of the vascular strand changes from circular to elliptical at a short distance below the bifurcation and eventually divides into two equal strands that continue into the axes above the dichotomy. All better-known species had apical meristems, resulting in a straight prolongation of the axes, except for Trichopherophyton teuchansii, which has circinate tips like the zosterophylls.

For detailed descriptions of and discussions on branching modes see:

Aglaophyton majus: [1,2,22]; Rhynia gwynne-vaughanii: [1,2,27,29]; Horneophyton lignieri: [2]; Nothia aphylla: [24–26]; Asteroxylon mackiei: [3]; Trichopherophyton lyonii: [16]; Ventarura lyonii: [17].

5. Emergences, spines and leaf-like structures

None of the Rhynie chert plants had real leaves. Aglaophyton majus, Horneophyton lignieri and Ventarura lyonii have smooth axes. The axes of Rhynia gwynne-vaughanii are basically smooth, but can look irregular due to the presence of small irregularly positioned bulk-like outgrowths, the so-called hemispherical projections. In some axial portions they can be very common, although plants with longer axial segments without hemispherical projections may occur.

The axes of Nothia aphylla have an irregular surface with short discontinuous and alternating longitudinal ribs formed by alternating lens-shaped stomata-bearing emergences and stomata-free depressions.

Asteroxylon mackiei had small leaf-like emergences or enations (figure 4a,e,f), which are, however, not vascularized are, therefore, cannot be termed leaves. To avoid unnecessarily long discussions on terminology, they are, following Kidston & Lang [3] and Lyon [8], here informally called ‘leaves’. Leaf traces given off by the central vascular strand run up to the periphery of the axis where they end abruptly just before the base of the leaves. The ends of the leaf traces are often marked by clumps of dark (?necrotic) cells (figure 4e), of which the function is unknown. Only the stalks of sporangia, which consist of two valves, are vascularized. The leaves are up to 5 mm long and do not show a regular phyllotaxy but they are more densely positioned in the upper parts of the upright axes.

Although Trichopherophyton teuchansii is comparatively poorly known, it is readily recognizable by the presence of rigid unicellular, sharply pointed spines, which are commonly 200–400 µm long but can occasionally be up to 1000 µm long. These spines are usually filled with a dark substance. Ventarura lyonii is easily identified by the strongly convex periclinal walls of the epidermal cells of which some bear up to 0.45 mm long protrusions with rounded tips, some of these are curved.

For detailed descriptions of and discussions on emergences, spines and leaf-like structures see:

Rhynia gwynne-vaughanii: [1,18,29]; Nothia aphylla: [24,25]; Asteroxylon mackiei: [3,30]; Trichopherophyton teuchansii: [16]; Ventarura lyonii: [17].

6. The anatomy of the aerial axes and rhizomes

If well preserved the following tissues can be recognized in the aerial axes, from the centre of the axis to the periphery: (i) the conducting strand consisting of thick-walled cells with or without wall thickenings, (ii) a parenchymatous tissue without intercellular spaces encircling the water-conducting strand, (iii) the cortex that is usually differentiated into an inner and outer cortex, but in Asteroxylon and Ventarura three cortical zones can be distinguished, (iv) if present, a hypodermis that varies from a single to up to four cell layers thick and (v) the outer cell layer or epidermis, which is overlain by (vi) a non-cellular layer, the cuticle (figure 1a–d). In some taxa also the anticlinal walls of the epidermal cells and the underlying hypodermis may be thickened (cutinized?) (figure 2b). None of the Rhynie chert plants possesses an endodermis. Ventarura has a mid-cortical zone consisting of sclerenchymatous cells.

(a). The conducting tissues

The conducting system of the aerial axes of all Rhynie chert plants consists of a central core of elongate thick-walled water-conducting cells with uniformly thickened walls like the hydroids of bryophytes, or tracheids with annular or spiral secondary wall thickenings. A tissue composed of elongate parenchymatous cells without intercellular spaces encloses this central core of thick-walled cells. Kidston & Lang [1] and several subsequent authors designated this tissue as phloem because it occupies the position of the phloem (e.g. [31]), although distinct sieve areas and the characteristic pitting have never been found in any of the Rhynie chert plants. These cells may closely resemble the leptoids of bryophytes [32].

(i). The water-conducting strand

The central strand of Aglaophyton majus consists of two differently sized cell types, a core of smaller, dark-coloured, thin-walled cells. The thin-walled cells in the centre of the strand are rather angular, four- to six-sided, isodiametric, 18–44 µm in diameter; up to seven successive cells can be counted from the centre to periphery of the inner part of the core (figure 2a). The inner core is surrounded by two to four, usually three layers of larger thick-walled, more elongate cells that are 22–50 µm in diameter in cross-section. The walls of the outer cells are 1.5–2.0 µm thick, double-layered, the inner layer being thicker. In rhizomes, a single strand of water-conducting cells may be present in the ventral part of the parenchymatous tissue encircling the water-conducting strand. Edwards [23] gave the most detailed description of the central strand of Aglaophyton and neither he nor Kidston & Lang [2] could find any evidence of secondary thickenings on the conducting cells. Hence, Edwards regarded the reticulate and hexagonal patterns on the walls reported by previous workers as being the result of (partial) enzymatic or biological degradation of the wall. Consequently, he considered the conducting strand of Aglaophyton to be more similar to the hydroids and leptoids of bryophytes, particularly larger modern Polytrichales, which instigated him to transfer the species to a new genus. In longitudinal sections, Edwards also noted the presence of spherical vesicles in the walls of the conducting cells. Careful observation shows that these may coalesce laterally and form a continuous layer, resembling the wall structures seen in Rhynia gwynne-vaughanii (figure 1e). This could mean that, A. majus is more closely related to R. gwynne-vaughanii than was suggested by Edwards [23]. Further studies, applying different techniques, may help to solve this problem and clear the systematic position of A. majus.

Rhynia gwynne-vaughanii has a vascular strand that is circular in cross-section. The proportion of the xylem is relatively small compared to that of the encircling parenchymatous tissue (figure 2d) and consists of fewer cells than in Aglaophyton (see below). In cross-section details of the xylem are often difficult to discern because the cells are very narrow and dark-coloured. In smaller axes all thick-walled cells composing the xylem are of uniform size. In larger axes of Rhynia the exarch xylem is differentiated into a central core of smaller cells surrounded by a zone of larger cells. In longitudinal section the tracheids show broad annular or helical thickenings (figure 1f). The tracheids of R. gwynne-vaughanii are of the S-type according to the classification of Kenrick & Crane [33] and Edwards [34,35].

The bulbous corms of Horneophyton lignieri do not contain conducting elements (figure 1g). The conducting strand starts at the base of the aerial axes, but there is a transition from the axial strand downward into files of dark cells without tracheidal thickenings in the tubers. The water-conducting strand is best preserved in the basal parts of the aerial axes. It is cylindrical, rather wide and exclusively composed of thick-walled cells. The central part is composed of smaller cells, but these are often not or very poorly preserved but a few specimens with better-preserved cells have been illustrated [2]. The central part is surrounded by a zone of up to eight layers of larger cells that gradually increase in size towards the periphery of the strand. In the longitudinal view, the cells of the central water-conducting strand often appear to be broken, whereas the surrounding more peripheral cells are still intact. The cells in the basal parts of the aerial axes show very narrow interconnected annular and helical thickenings (figure 1h).

The vascular core of aerial axes of Nothia aphylla is usually poorly preserved; this in contrast to the horizontal rhizomes (see below). In the latter, the strand of water-conducting cells varies from circular to slightly elliptical in cross-section and shows a differentiation into smaller cells (approx. 10 µm in diameter) in the centre and an outer zone of larger cells (<30 µm in diameter). The rhizomes of Nothia consist of laterally branched systems comprising several orders of branching. The horizontal, rhizoid-bearing axes and the basal vertical portions of the upright axes are much better preserved than the aerial axes and show a great amount of detail. The horizontal axes are dorsiventrally symmetrical in cross-section and have a more or less circular to slightly ventrally elongated vascular core that is 10–20 cells in number, 300–500 µm in diameter. Four different cell types can be distinguished, i.e. long-fusiform medullary cells with strongly thickened walls and tapering ends, thick- and thin-walled short cells arranged in longitudinal rows, which are interpreted as vascular parenchyma, and the phloem (see below). The long-fusiform cells are over 700 µm long and 10–35 µm in diameter. In their shape and the uniformly thickened walls, these cells resemble fibres or hydroids rather than tracheids. Small files of thick-walled cells extend from the vascular core downward and increase in size in the direction of the rhizoidal ridges; groups of thick-walled cells or single cells also occur isolated, enclosed by phloem. Cross-sections reveal that the central core of water-conducting cells is very variable in shape where the vertical branches are attached.

Asteroxylon mackiei is the most complex of all Rhynie chert plants. This is also expressed in the histology of the vascular system in the several types of axes that can be distinguished, i.e. rhizome (horizontal) axes which give off upright stems and root-like axes. This differentiation has nicely been documented [3]. In cross-section, the rhizome axes that can be up to 6 mm in diameter show a vascular core with a simple more or less circular to elliptical strand of undifferentiated tracheids surrounded by a rather broad zone of phloem. All tracheids have spiral wall thickenings. In the transition to the upright axes, the outline of xylem strand changes gradually first to triradiate and cross-shaped in the lower parts of the upright axes (figure 4b) and in smaller axes, and eventually to the typical stellate outline in the larger upright leafy stems. The vascular strand of the upright stem is an actinostele. The xylem strand of the upright stems contains small groups of small cells representing the protoxylem. They are located close to the periphery of the xylem strand and the ends of the arms. The metaxylem that constitutes most of the xylem consists of larger cells. The tracheids of Asteroxylon are of the G-type; in the longitudinal view, the tracheids show annular wall thickenings (figure 4c). The central strand gives off leaf traces that can be recognized as small clusters of dark cells embedded in the encircling parenchymatous tissue and in the cortex more towards the periphery of the stem. In rooting axes, the xylem is triangular in cross-section and surrounded by a broad zone of parenchymatous cells without intercellular spaces.

The xylem of Trichopherophyton teuchansii is slightly elliptical in cross-section and the wall thickenings of the tracheids are annular or form a narrow spiral. Maturation was apparently centripetal; the larger metaxylem elements in the centre of the strand are 40–50 µm in diameter.

Ventarura lyonii has a cylindrical, probably exarch, xylem strand, 640–1860 µm in cross-section, composed of G-type tracheids with annular and spiral wall thickenings, arranged in an uneven but regular pattern. However, cells in the centre of medullary strands lack secondary thickenings. Tracheids are slightly tapering with rounded end walls, isodiametric, 11–42 µm in cross-section, and the shortest ones are 960 µm long; specimens with anomalous branching have much shorter tracheids.

For detailed descriptions of and discussions on the water-conducting system see:

Aglaophyton majus: [1,2,21,22]; Rhynia gwynne-vaughanii: [1,2,33]; Horneophyton lignieri: [2]; Nothia aphylla: [23,24]; Asteroxylon mackiei: [3,30]; Trichopherophyton teuchansii: [16]; Ventarura lyonii: [17]; General overviews: [34–36].

(ii). Parenchymatous tissues encircling the water-conducting strand

Rhynia gwynne-vaughanii has a four to five cells wide circular zone around the xylem, consisting of lighter coloured, tightly packed, thin-walled, four- to six-sided cells without intercellular spaces (figure 1a,b). This tissue is clearly separated from the surrounding inner cortex, which consists of more loosely arranged cells with prominent intercellular spaces. Moreover, in longitudinal section, these cells are much longer than the cortical cells and they have oblique instead of transverse end walls. The cells in the interior part are smaller than other cells, but they rapidly increase in size towards the outer part of the zone where they may reach the size of the adjacent cortical cells. The same can be seen in Aglaophyton majus in which this zone of parenchymatous elongated cells with oblique, S-shaped end walls, and lacking intercellular spaces, can in cross-section look less regular. At places this tissue may extend deeper into the cortex forming short rays. This tissue may then be up to eight cells wide and then has a vaguely, irregular star-shaped outline (figure 1b,c). Lines of cortical cells that are slightly larger than the other cortical cells form the continuation of the radiating cell rows of this tissue.

In primary subterranean axes of Nothia aphylla, the tissue connecting the rhizoidal ridges with the core of thick-walled water-conducting cells consists of tightly packed, thin-walled parenchymatous cells without intercellular spaces (figure 3b). These latter cells differ from the cells of the adjacent cortex in size and shape. The aerial axes (figure 3c) have fusiform, thin-walled cells with tapering or S-shaped ends that vary from 300 to over 700 µm in length and are 15–45 µm in diameter. They are not arranged in longitudinal files. Distinct sieve areas have not been observed.

The fine details of the aerial axes of Horneophyton lignieri are often less well preserved than in the above species and tissues with thin-walled cells are usually collapsed. In longitudinal sections, cells in the zone immediately adjoining the central strand of thick-walled water-conducting cells seem to be narrower and longer than the cells of the cortex [2].

A tissue composed of parenchymatous cells without intercellular spaces is usually well recognizable in the rhizomes and rooting axes of Asteroxylon mackiei, where it forms a broad zone encircling the xylem. In some axes, the xylem is located in an eccentric position; it is unclear whether this is a matter of preservation. The parenchymatous cells encircling the xylem are elongated and have transverse or pointed end walls. In the upright leafy stems, the xylem is always less well preserved. In these axes the outline of the encircling parenchymatous tissue is circular to elliptical in cross-section, just a few cells wide at ends of the xylem rays and fills up the spaces between the rays. The cells directly adjacent to the xylem are smaller than those filling up the spaces between the rays.

In Trichopherophyton teuchansii, a zone of cells (approx. 50–100 µm × 40–60 µm) without intercellular spaces encloses the xylem. Also in Ventarura lyonii, a zone of thin-walled cells lacking intercellular spaces, surrounding the xylem strand can be recognized.

For detailed descriptions of and discussions on the parenchymatous tissues encircling the water-conducting strand see:

Aglaophyton majus: [2,22,23]; Rhynia gwynne-vaughanii: [1,2]; Horneophyton lignieri: [2]; Nothia aphylla: [25]; Asteroxylon mackiei: [3]; Trichopherophyton teuchansii: [16]; Ventarura lyonii: [17].

(b). Cortex

The cortex consists of loosely packed cells, with often quite large intercellular spaces, which easily differentiate it from the adjoining parenchymatous tissue encircling the water-conducting strand, hypodermis and/or epidermis. Although the cortex normally constitutes most of the axial tissues, it has received only little attention so far. This may partly be because this tissue of thin-walled cells has, depending on the species, often been subject to severe decay and is then poorly preserved. The only exception is the middle cortex of Ventarura lyonii that consists of thick-walled cells.

In cross-section, larger axes of Aglaophyton majus and Rhynia gwynne-vaughanii show a clear differentiation into an inner and outer cortex (figure 1a–d). In smaller axes, this differentiation is less clear. The cells of the inner cortex are generally smaller than those of the outer cortex. The latter may (partly) be hypodermal in nature, although gradual transitions exist. The cell size within the inner cortex varies and larger cells may be up four times bigger than the smaller ones, but cells generally increase in size towards the outer periphery of the inner cortex. Cells are arranged in files radiating from the centre of the axis. This is particularly clear when (bundles of) files of larger cells alternate with files of smaller cells. Cells are rounded to sub-angular in cross-section and higher than wide in the longitudinal view. Intercellular spaces are prominent and can be fairly large. The cells of the outer cortex are larger and more angular in cross-section, and intercellular spaces are much smaller. In the longitudinal view, the cells of the outer cortex are wider than high, except for the outer layer. In Aglaophyton, the outer cortex is up to five (usually three to four layers, in smaller axes even less) and in Rhynia only one or two cell layers thick. In both species, a prominent dark ring marks the transition from the inner to the outer cortex. This dark colour is caused by the presence of hyphae and vesicles of mycorrhizal fungi often completely filling up in the intercellular spaces of the outermost layer(s) of the inner cortex.

The rhizomes of Horneophyton lignieri consist of parenchymatous cells that are aligned in files with small intercellular spaces. The intercellular spaces become smaller towards the periphery of the tubers. The outer cortex of the aerial axes consists of three to five cell layers of more or less isodiametric cells (50–150 µm in diameter) with large intercellular spaces that are larger in the vicinity of the stomata. The inner cortex is composed of longitudinally elongated cells (150–170 µm long and 30–50 µm wide) with truncated or overlapping end walls. The cell length gradually decreases towards the outer region of the inner cortex and intercellular spaces become smaller towards the central region of the axis.

In rhizomes of Nothia aphylla, the cortex is horseshoe-shaped and ventrally interrupted by the connective that connects the conductive strand with the rhizoidal ridges (figure 3b). The cortical cells are elongate (100–300 µm long and 50–85 µm wide), rounded in cross-section with rounded or tapering end walls forming longitudinal files and large intercellular spaces. The aerial axes show a clear pattern of emergences and depressions (for further details, see 6(c) Dermal structures). This differentiation is also visible in the outer cortex. Cortical cells underlying the depressions are small (60–80 µm long and 60–80 µm deep). In some cases, they are radially extended and up to two times deeper than long. The end walls are oblique and the cells make an angle of approximately 60° with the hypodermis. The outer cortex under the emergences is very spongy due to the presence of very large intercellular spaces. Cells can be spherical to cylindrical and 70 µm in diameter, often with protrusions. Also up to 120 µm deep and 50–60 µm long cells may occur. The inner cortex is formed by elongate cells, which increase in length and become about twice as long in the inner part of the cortex, where intercellular spaces are much smaller.

The rhizomes of Asteroxylon mackiei show a differentiation into an inner and an outer cortex that is clearly marked by differences in colour and cell size. The inner cortex consists of thin-walled small, cells, which are usually very poorly preserved. The cells are sub-angular to rounded in cross-section and slightly elongate in the longitudinal view. The cells of the outer cortex are similarly shaped but larger. The outer cortex consists of two to five cell layers; in specimens where five layers are present the cell size increases towards the periphery. The cortex of the upright leafy shoot is always poorly preserved, but it is also differentiated into an inner and an outer cortex; no details of the latter can be given, because it usually largely absent due to decay. The inner cortex may be uniformly developed but is usually differentiated in three zones. The middle zone, which is the thickest and most prominent, is characterized by the arrangement of cells in radiating strands separated by wide intercellular spaces. The narrower inner and outer zones of the inner cortex are less conspicuous.

Trichopherophyton teuchansii has an inner cortex with loosely positioned isodiametric, thin-walled cells. They are 40–50 µm is diameter in cross-section and more or less arranged in radiating rows. The outer cortex is narrower, more compact and intercellular spaces are smaller.

In Ventarura lyonii, the cortex is divided into three zones. The inner cortex with its very large intercellular spaces is often poorly preserved and consists of (sub)rounded thin-walled cells that are 27–52 µm in diameter in cross-section and slightly elongate in the longitudinal section. The middle cortex consists of up to seven but usually less layers of thick-walled cells. The cell diameter is similar to that of the inner cortical cells, but cells may be slightly elongated radially and become smaller in the outer part of this zone. The length of these cells with transverse to oblique end walls may be at least 700 µm. Also the outer cortex is usually poorly preserved and consists of closely packed, very thin-walled cells with few intercellular spaces.

For detailed descriptions of and discussions on the cortex see:

Aglaophyton majus: [2]; Rhynia gwynne-vaughanii: [1,2]; Horneophyton lignieri: [36]; Nothia aphylla: [24,25]; Asteroxylon mackiei: [3]; Trichopherophyton teuchansii: [16]; Ventarura lyonii: [17].

(c). Dermal structures: epidermis, hypodermis, glands, rhizoids and the cuticle

All species have a single-layered epidermis, except for Nothia aphylla, which, between the giant cells, may have two layers of epidermal cells. A hypodermis is present in Aglaophyton majus, Rhynia gwynne-vaughanii, Horneophyton lignieri and Nothia aphylla. Glandular structures have been described from Horneophyton. All taxa except for Asteroxylon mackiei have rhizoids. Stomata are always simple and lack subsidiary cells; the neighbouring cells generally resemble the other epidermal cells.

The epidermis of the aerial axes of Aglaophyton majus shows a large variation in cell shape, cell size and cell arrangement. Cells can be up to 350–800 µm long in the rhizomes with oblique or transverse end walls, but they reach about half the length in the upright axes. Just below the bifurcations and the attachment of the sporangia, the epidermal cells are only 150–200 µm long and arranged in horizontally or spirally arranged regular blocks. The epidermis is underlain by a one or two cell layers thick hypodermis (figure 2b). The cells of the epidermis and hypodermal layer(s) are narrower than those of the underlying cortex. Stomata (120–140 µm long and 85–100 µm wide) are longitudinally oriented, rather common on the axes and less common and smaller on the sporangia and in the rhizomes. The 7–11 neighbouring cells are similar to other epidermal cells but usually shorter. Stomata are not sunken, elliptical in outline and the substomatal chamber may reach up to five cell layers below the surface (figure 2c). The walls of the hypodermal cells facing the narrow channel leading from the stomatal pore to the substomatal pore are cutinized. In older axes, the cutinization extends to the hypodermis. The guard cells of the stomata on the aerial axes are usually filled with dark-coloured substance (figure 2e), whereas those for the stomata on the rhizomes have the same colour as the neighbouring epidermal cells. The reniform guard cells bear stomatal ledges and have more or less triangular outgrowths in the polar regions. The cuticle covering the epidermis is up to 5 µm thick and some cells show median cuticular ridges as can be seen in cross-sections of the cuticle.

Most typical for axes of Rhynia gwynne-vaughanii is the common occurrence of small bulge-like outgrowths, the so-called hemispherical projections (figure 3a). These will be described in a following subsection. The epidermis of the full-grown aerial axes is composed of randomly distributed shorter and longer cells that vary in length from 130 to 450 µm and are 10–30 µm wide; epidermal cells of thinner axes are smaller. Stomata occur on the aerial axes including the hemispherical projections but have, unlike in Aglaophyton majus, not been found on the sporangia. The up to 4 µm thick cuticle of Rhynia frequently shows median ridges. These have been interpreted as the result of post-mortem shrinking, however, cross-sections of Rhynia cuticles reveal that the cuticle of some epidermal cells shows a central thickening. Similar ridges are also found on the epidermal cells of Nothia and Asteroxylon. The stomata of Rhynia are oval to almost circular in outline, 90–100 µm long and 75–85 µm wide, and not sunken. The seven to nine neighbouring cells resemble the other epidermal cells in size and shape. The channel leading to the substomatal chamber is less well developed than in Aglaophyton and the adjoining hypodermal cells are not cutinized (figure 3f). The hypodermis is usually a single cell layer thick.

The epidermis of the basal corm of Horneophyton lignieri consists of regular patches of up to 16 rectangular to quadrangular cells on dorsal and lateral sides and less regularly shaped cells on the ventral side. The epidermis of the axes consists of elongate cells that can be up to 1000 µm long with tapering ends, which become shorter (600 µm) towards the rhizomes. The stomata are scattered, vary considerably in size (120–180 µm long and 70–115 µm wide), and are enclosed by four to seven neighbouring cells that are similar to other epidermal cells; the reniform guard cells with prominent stomatal ledges. The epidermis is underlain by a one cell-layer thick hypodermis. Scattered glandular structures consist of an opening surrounded by two rings of small non-cutinized cells. The up to eight neighbouring epidermal cells are shorter than the other epidermal cells. The epidermis of the sporangium wall consists of elongate cells up to 500 µm long with straight lateral and slightly sinuous end walls. Towards the tip of the sporangium the cell length decreases gradually until cells are approximately 100 µm long shortly below and finally isodiametric, 50–60 µm in diameter, at the apex. Stomata are scattered, circular in outline and smaller than those of the aerial axes.

The dermal tissues of Nothia aphylla show a complex pattern and a large variation of cell shapes and sizes, unlike any of the other Rhynie chert plants. The epidermal cells on the lateral sides of the rhizomes are elongate and up to 200 µm long, whereas those on the ventral rhizoidal ridges are short, 30–50 µm long, 35–50 µm wide. They are 35–60 µm deep in the lateral parts only 15–35 µm deep in the central parts of the rhizoidal ridges. Almost every cell of the central part of the rhizoidal ridge bears an unbranched up to 100 µm long rhizoid. Younger (shorter) rhizoids are may have tapering tips, whereas older ones have a slightly swollen tip. The epidermis of the rhizoidal ridges overlies a three- to seven-cell–layered thick hypodermis, which lacks intercellular spaces. In many cases the hypodermis shows a threefold subdivision expressed in different cell sizes. The cells of the outer layers are short and shallow, 20–50 µm long, 50–105 µm wide and 50–80 µm wide, whereas those of the middle layer are equally wide but 35–80 µm long and 120 µm to over 200 µm deep. The cells of the inner hypodermal layer adjacent to the connective layers are more or less cuboidal and 70–120 µm long, 70–85 µm wide and 60–120 µm deep. The epidermis of the mature aerial axes shows an alternation of longitudinally oriented cells, giant cells, files of short cells and more less spirally arranged stomata-bearing emergences. The giant cells are up to 1.6-mm long, 90–110 µm wide in the longitudinal view and up to 200 µm deep in cross-section. These giant cells occur isolated and appear very narrow in the surface view (10–20 µm or less) because they are largely obscured by (partly) overlying short epidermal cells. The walls of the giant cells are not thickened; this in contrast to the short cells that always have strongly thickened anticlinal and periclinal walls. The number of giant cells decreases towards the base of the aerial axis. The short epidermal cells are 120–300 µm long, 15–100 µm wide in the longitudinal view and 30–80 µm deep in cross-section and have transverse or oblique end walls. Shorter and longer short cells occur irregularly within single-cell files. These may comprise up to 15 cells reaching a total length of more than 2.5 mm. The short cells adjacent to the giant cells are wider than those at some distance from the giant cells. The giant cells alternate with two to seven (sometimes up to 12) files of short cells. The aerial axes show conspicuous 250–350 µm high elongate emergences. In the surface view, the longitudinally oriented emergences are elliptical to lens-shaped, 700–1200 µm long (400–600 µm near the bifurcations) and 300–500 µm wide. The stomata-bearing emergences consist of 7–10 files of short cells and 2–4 intervening giant cells. The short cells in the depressions between the emergences are narrower (10–30 µm wide) than those of the emergences (50–100 µm wide). Emergences can be densely positioned and form longitudinal ridges. All anticlinal and periclinal walls of the epidermal cells are strongly thickened, except for the giant cells. A second epidermal layer of equally shaped, sized and thickened cells underlies the outer epidermal layer. Also this layer is interrupted by giant cells. This tissue forms a double-layered epidermis. The stomata, which are positioned on the summits of the emergences on the aerial axis and sporangia, are anomocytic and almost circular in outline (75–105 µm long and 70–95 µm wide). The six to eight neighbouring cells resemble the normal epidermal cells in size and shape, although the lateral ones cells can be very short. The stomata are not sunken and the reniform guard cells are almost rectangular in cross-section with very thick periclinal walls, primarily the outer but sometimes also the inner periclinal walls.

The epidermis of the rhizomes, the aerial axes, the leaf bases and the leaves of Asteroxylon mackiei consist of isodiametric polygonal cells and slightly elongate cells that are up to twice as long (75–150 µm) as wide and have oblique or transverse end walls. The periclinal walls are thickened; they are smooth or they may bear papillae. Sometimes the anticlinal walls show median ridges as in Rhynia. Towards the root-like axis the cells become longer and are up to four times longer than wide. The epidermal cells of the root-like axis are longitudinally oriented and occur in short files of two to four cells; the outer cells within a file have strongly tapering end walls, whereas the end walls of the cells within a file are transverse to slightly oblique. Stomata are densely positioned and occur randomly on the rhizomes and the aerial axes, but they are lacking on the root-like axes, the leaves and the sporangia. They are sunken, mostly circular in outline, 50–70 µm long and 50–65 µm wide. The reniform guard cells are filled with a dark substance and bear stomatal ledges (figure 4d). The neighbouring cells generally resemble the normal epidermal cells, but they often radiate from the stoma in a more or less rosette-like arrangement.

Trichopherophyton teuchansii and Ventarura lyonii lack a hypodermis. The four- to six-sided, epidermal cells are longitudinally elongated. Those of Trichopherophyton may show rounded hair bases. Stomata have not been observed in these two species.

For detailed descriptions of and discussions on dermal structures see:

Aglaophyton majus: [1,2,22,27,36,37]; Rhynia gwynne-vaughanii: [1,2,36,37]; Horneophyton lignieri: [36,38]; Nothia aphylla: [24,25,36,37]; Asteroxylon mackiei: [3,36,37]; Trichopherophyton teuchansii: [16]; Ventarura lyonii: [17].

7. Sporangia and in situ spores

Several types of sporangia are found in Rhynie chert plants. The place and mode of attachment as well as the dehiscence mechanism differs among the different species. Aglaophyton majus and Rhynia gwynne-vaughanii both have elongate, spindle-shaped sporangia with rather thick walls that are terminally attached. Horneophyton lignieri has terminally attached branched sporangia with a central columella. Sporangia of Nothia aphylla and Asteroxylon mackiei are stalked, laterally attached and consist of, or open into two valves. All Rhynie chert plants were homosporous.

Sporangia of Aglaophyton majus are common. The most detailed description of the sporangia of A. majus is given by Kidston & Lang [1]. However, they initially attributed them—mainly on the basis of association—to Rhynia gwynne-vaughanii. Later they realized that the plant they originally described as R. gwynne-vaughanii includes two different species, a plant with the larger axes and one with the smaller axes. The one with larger axes, lacking adventitious branches and hemispherical projections was named Rhynia majus [2]. This species was later transferred to Aglaophyton [23]. Sporangia of Aglaophyton are terminally attached to aerial axes. The spindle-shaped sporangia with a rounded apex are large, larger than the sporangia of any other Rhynie chert plant; they are up to 12 mm long and 2.5 mm wide. The sporangium wall is up to 0.4 mm thick and consists of several layers, an outer epidermal layer covered by the cuticle, a layer of parenchymatous cells and an inner tapetal layer. In axes bearing the mature or emptied sporangia, the axes contracted just below the base of the sporangium where the vascular strand ends. The large number of isolated sporangia in the chert supports the interpretation that this was an abscission zone. The sporangia opened via a longitudinal dehiscence slit or an oblique dehiscence slit forming a very lax spiral. Wellman et al. [39] calculated that a single sporangium could have contained approximately 400 000 mature single spores. The spores are trilete and both the proximal and distal surfaces are laevigate. The proximal surface shows a distinct thinning associated with the trilete mark. The spores are up to 85 µm in diameter, but most are around 65 µm. They have been identified as Retusotriletes sp. [39].

Rhynia gwynne-vaughanii axes with attached sporangia are extremely rare. The sporangia originally attributed to R. gwynne-vaughanii [1] later appeared to belong to Aglaophyton majus [2]. Given their rareness, it is not surprising that definite proof of the sporophytic nature of Rhynia gwynne-vaughanii was not given until 1980 [27]. The axes of Rhynia branch more often than those of Aglaophyton. Sporangia are terminally attached, but overtopping adventitious branches occur just below the attachment of the sporangium. The thick-walled sporangia are spindle shaped, up to 2.4 mm wide and 5.1 mm long but usually shorter. The sporangium wall consists of three layers, an epidermis without stomata, a middle layer of parenchymatous cells and an inner tapetum. No clear dehiscence mechanism has been recognized, although some sporangia show a longitudinal slit near their apex. The vascular strand widens near the base of the sporangium and tracheids become shorter. In some sporangia a short, up to 0.4 mm high sterile pad composed of short tracheidal cells and parenchymatous cells is present at the base of the sporangium. Empty sporangia often show a black layer at the base, which has been interpreted as an abscission layer, although a clear narrowing of the xylem strand is absent. The trilete in situ spores with an apiculate outer layer are up to 53 µm in diameter (mean diameter 39 µm) and have been identified as Apiculiretusispora plicata [40].

In Horneophyton lignieri, sporangia are attached to the tips of some branches. Eggert [41] provided the most detailed descriptions of the sporangia of Horneophyton. The sporangia are branched into usually two lobes but sometimes into four lobes, forming a single sporangial cavity. Each lobe is served by a well-developed vascular strand, which bifurcates at a short distance below the basis of the sporangium, but does not continue into the sporangium. The sporangium lobes are more or less globose to cylindrical, thick-walled with a prominent central column of sterile tissue that occupies half to up to two-thirds of the length of the sporangium (figure 3f). The resulting sporangial cavity is inverted cup–shaped and shows a dark-coloured tapetum layer at the inner side of the sporangium. No clear dehiscence slits have been observed and it has been suggested that the sporangia opened via a central pore at the truncated apex of the sporangium. A single sporangium may contain a mixture of tetrads (figure 3g) and mature spores. Most remarkable is the presence of a fair number of dyads, decussate tetrads (notably ring tetrads) and aborted tetrads. The latter consist of large spore mother cells showing incomplete meiosis with only partially developed proximal spore walls. Mature normal spores are 39–45 µm in diameter with a well-developed trilete mark, which extends into curvaturae perfectae. The proximal surface bears 12–21, usually prominently developed, radial ribs. The distal surface has an apiculate ornament consisting of irregularly distributed, <0.5–1.5 µm high granae, coni and spinae. These spores are of the Emphanisporites decoratus type, a dispersed spore taxon that is often abundant in the polygonalis-emsiensis Zone (late Pragian–early Emsian) and is geographically widespread, with occurrences in Western Europe, Svalbard and Canada [40].

In Nothia aphylla, sporangia are laterally attached and borne on a short, 1–4 mm long, more or less perpendicularly inserted, adaxially recurved stalk, each bearing a single reniform sporangium, approximately 1.5 mm long, 3 mm wide and 1.2 mm thick; sometimes two connected stalks bear two fused sporangia. The sporangium stalk tapers gradually towards the point of attachment of the sporangium, where dark (necrotic?) cells occur. It is unclear whether this is an abscission zone, because it looks very similar to the sporangiophores in Asteroxylon, although isolated stalks from which the sporangia have been abscised have never been found. The long axis of the sporangium is usually horizontal with the stalk inserted more or less in the middle of the long (outer) side; in some sporangia the long axis is oblique or vertical. The sporangia walls are up to five cell layers thick and the long transverse dehiscence slit occupies most of the sporangium length and faces the main axis. The dehiscence slit is slightly widened at both ends and surrounded by annulus-like cells with thickened walls. The two valves resulting after dehiscence are slightly unequal in size, the upper one being a little bit longer. Sporangia may either occur at random, be spirally arranged on the axis, or be standing close together in pairs borne at the same level or be forming whorls of three, or they may be radially arranged to form terminal clusters consisting of up to five sporangia. Some axes only partially bear sporangia. El-Saadawy & Lacey [24] questioned whether such axes were indeed only partially fertile, and fertile and sterile portions were alternating. Regarding the same phenomenon occurring in Asteroxylon [30], this is not unlikely. According to Wellman [40] the in situ spores of Nothia aphylla that are up to 65 µm in diameter can be identified as Retusotriletes sp.

In Asteroxylon mackiei, the fertile axes (figure 4e,f) vary in width from 8.9 to 2.0 mm at the apex. Sterile axis segments alternate with fertile intervals. In the fertile portions of the stem, the sporangia are randomly positioned between the leaf-like appendages. There is no correlation between the number of leaf-like appendages and the number of sporangia. Sporangia are attached to the main axis with a short stout stalk with an angle of 45°. The sporangia are kidney shaped, 1.7–15.2 mm long and 0.6–5.6 mm wide, and consist of two valves (figure 4f,g) of which the outer one is usually strongly convex, whereas the inner valve that is faced towards the axis is flat or concave. The sporangia are curved and lie parallel to the periphery of the axis. The stalk is positioned centrally and continues into the proximal part of the sporangium where it forms a dome-shaped structure (sterile pad) that connects both valves and probably extends up to one-third of the sporangium length. The vascular bundle of the main axis gives off a vascular strand, which enters the sporangium stalk, narrows where it enters the sporangium, and then bifurcates to enter the valves and continue into the sterile pad. Within the sterile pad the tracheids are short radiate. The vascular strands continuing in both valves directly underlie the tapetum and gradually taper towards the apices of the valves. The sporangia have a distinctive dehiscence structure extending along almost their entire circumference and consisting of an elevated rim and a groove in each valve. The conspicuous narrowing of the vascular strand at the base of the sporangium, the dark-coloured necrotic cells in this region and the presence of short stalks not (longer) bearing sporangia indicate the existence of an abscission mechanism. Indeed, several isolated sporangia have been found. The very simple retusoid trilete spores that are 44–59 µm in diameter show a thickening on the proximal side associated with the trilete mark. These spores have been identified as Retusotriletes cf. triangulatus.

The sporangia of Trichopheropyhton teuchansii and Ventarura lyonii are both attached laterally and consist of two unequally thick, kidney-shaped valves. Only very few specimens of Trichopherophyton with attached sporangia are known. Sporangia were sessile, standing erect and 2.3–2.5 mm high and 3.0–3.75 mm wide. It is still unclear whether sporangia were inserted in strobili or not. The vascular strand ends in the abaxial valve that is thicker than the adaxial one. Sporangia of Ventarura are borne in strobili, attached with a short, stout stalk and positioned more or less perpendicularly to the stem. They are up to 4 mm long, 5.2 mm wide and 2 mm thick. The vascular strand continues in the basal third of the abaxial valve that is thicker than the adaxial one. A zone of dark cells is interpreted as the abscission tissue. In both taxa dehiscence took place via a long slit running along the convex distal side of the sporangium.

For detailed descriptions of and discussions on sporangia and in situ spores see:

Aglaophyton majus: ([1]: described as Rhynia gwynne-vaughanii, [39,42,43]); Rhynia gwynne-vaughanii: [27,40]; Horneophyton lignieri: [2,40,41,44,45]; Nothia aphylla: [24,40]; Asteroxylon mackiei: [8,30]; Trichopherophyton teuchansii: [16]; Ventarura lyonii: [17].

8. Gametophytes, gametangiophores and gametangia

Regarding the exquisite preservation in the Rhynie chert, at least in specific facies, the presence of the gametophytic generation of the well-known sporophytes could be expected. During several decades speculations arose about supposed gametophytes (e.g. [46–48]). Probably because sporangia of Rhynia gwynne-vaughanii remained unknown for 60 years, several authors misinterpreted irregular structures on the axes of Rhynia gwynne-vaughanii, such as hemispherical projections and obliquely cut stomata, as gametangia. They speculated that R. gwynne-vaughanii would be the gametophyte of Aglaophyton majus, but when Edwards [27] provided the first description of the sporangia, the sporophytic nature of R. gwynne-vaughanii was proved. In the same year Remy & Remy reported an antheridia-bearing gametophyte under the name Lyonophyton rhyniense [10,11]. In later years, several other gametophytes were described from the Rhynie chert. The gametophytes were free-growing non-thalloid plants, their vegetative parts resembling the sporophytes, but they were much smaller. In all species, gametophytic axes consist of a well-developed conducting strand, a cortex, an epidermis and a cuticle with stomata, and archegonia or antheridia positioned near or at the apex of the axis. The correlations between sporophytes and gametophytes (table 1) are based on anatomical similarities, notably in the conducting tissues and the structure of the axial surface. These correlations are further substantiated by close co-occurrences in the same biofacies type. Although they are isomorphic, they differ in size, not only in overall size but also in the size of the individual cells, including the stomata, which are smaller. Of at least two species for which several developmental stages are known, the alternation of generations is now known in great detail [15,49].

Table 1.

Sporophytes and gametophytes of Rhynie chert plants.

sporophytes	gametophytes
Aglaophyton majus	Lyonophyton rhyniense
	♀	♂
Rhynia gwynne-vaughanii	Remyophyton delicatum
	♀	♂
Horneophyton lignieri	Langiophyton mackiei
	♀	♂
Nothia aphylla	Kidstonophyton discoides
	?	♂

Lyon reported the first gametophytes when he described germinating spores from the Rhynie chert [50]. Bhutta described additional germinating spores from the chert, including initial developmental stages of germinating Horneophyton lignieri spores with first cell divisions [42,44]. However, later developmental stages and gametangia remained unknown until Remy & Remy described the first structurally preserved antheridia-bearing gametophyte as Lyonophyton rhyniense [10,11]. This appeared to be the gametophyte of Aglaophyton majus. In later years, additional descriptions were published, and several other gametophytes were described, respectively, as Langiophyton mackiei [13,15], Kidstonophyton discoides [14] and Remyophyton delicatum [15], respectively, belonging to Horneophyton lignieri, Nothia aphylla and Rhynie gwynne-vaughanii. In Kidstonophyton only gametangia-bearing axes are known. Most gametophytes are incomplete and usually only the terminal parts of the gametangia-bearing axes are known, except for Remyophyton delicatum that is preserved as a monospecific in situ stand of more than 200 complete unisexual individuals. Also of L. rhyniense the basal parts of the axes are known. Lyonophyton gametophytes were obviously also unisexual, because the axes either bear antheridia or archegonia. The gametophytes of Asteroxylon, Trichopherophyton and Ventarura are still unknown. Asteroxylon grew on better-drained substrates where the preservation potential is very limited. The other two species are extremely rare and there are not even whole-plant reconstructions of the sporophytes.

The antheridia-bearing axes of Lyonophyton rhyniense, the gametophyte of Aglaophyton majus, are at least 2 cm long and 2 mm wide (figure 5c). They end in a shield- to bowl-shaped structure. Small antheridiophores are entire-margined, whereas larger bowl-shaped ones have marginal lobes. The lobes are thick and fleshy and stand up. The antheridia are borne on the upper part of the shield, respectively, in the interior of the bowl and the lobes. Antheridia (figure 5d,f) are attached with a short stalk and more or less spherical, sometimes kidney-shaped; they are more or less pear-shaped when mature. They have with a central column-like structure of sterile tissue at the base that may reach up to one-half of the antheridium height. The antheridium wall is one or two cell layers thick and the antheridia have an apical operculum. Sperm cells in immature antheridia are coiled and arranged in rows radiating from the centre and in individual packets. In between there are thin strands of parenchymatous cells diverging from the central column to the antheridium wall. The archegonia-bearing axes are dichotomously branched and have a flattened apex. Archegonia (figure 5g) are standing isolated on the apex, subapically and laterally just below the apex. The archegonia are nearly hemispherical with a thick neck, a neck canal and a deeply sunken egg chamber. The sterile tissues of the gametophytes are similar to those of the sporophyte, with a massive conducting strand of tracheid-like cells with bubble-like bodies forming clusters and chains surrounded by a zone of thin-walled cells without intercellular spaces, a massive cortex, a hypodermis, an epidermis and a cuticle with stomata.

The characterization of the gametophyte Lyonophyton rhyniense given above is based on over 150 antheridia-bearing axes. However, archegonia-bearing ones are less common. Together this rich material enabled the reconstruction of the alternation of generations in an early land plant [49]. Germinating Aglaophyton spores are not uncommon in the Rhynie chert (figure 5a). They are primarily bound to humid and wet facies, e.g. on microbial and Palaeonitella mats. Once they landed on a favourable substrate, the spores started to swell, opened along the trilete mark and a globular mass extruded. In some cases, the spore wall is ruptured and only shreds of the spore wall are still visible. The first cell division was transverse and the outer cell continues to divide, as does the cell inside the spore wall. After a series of cell divisions (figure 5b) described in detail [49], the young gametophyte grew out to a teardrop-shaped structure. In the gametophyte that will eventually give rise to the antheridia-bearing axes, the apical part expanded laterally to form a dish-like protocorm lying on the substrate. Unicellular rhizoids developed on the lower side of the protocorm and vertical antheridia-bearing axes departed from the upper side. A dichotomously branched archegonia-bearing axis developed from the young teardrop-shaped gametophyte, but it is still unclear whether there was a well-defined protocorm stage.

Remyophyton delicatum is known from a single monospecific in situ stand of over 200 individuals, all arising from a 1.5 × 1.5 cm patch on a sinter surface with filamentous microorganisms (figure 5h). Each gametophyte consists of a single unbranched axis; the ones in the centre of the stand are erect, whereas the marginal ones, which are more loosely positioned than those in the centre, are more or less prostrate. In several cases the remnants of the spore wall are still visible at the basis, as are rhizoids. The individual axes are of different length. The larger antheridia-bearing axes are usually 10–15 mm long but some may some reach up to 20 mm, whereas the smaller are only 4–6 mm long and bear the archegonia. The parenchymatous, rhizoid-bearing protocorm is globular or bulbous, irregularly lobed or slightly bowl-shaped. Stomata may occur on the upper surface of the up to 1 mm high protocorm. A small incision marks the boundary between the protocorm and the aerial axis. All tissues resemble those of the young axes of Rhynia gwynne-vaughanii, but the cells are smaller. Tracheids appear about 1 mm above the base of the aerial axis. The axes may be slightly widened at the apex. The antheridia are more or less globular and attached with a short stalk to the flattened apex or laterally just below the apex. Axes usually bear one to three antheridia but some specimens as much as five. The archegonia are positioned laterally and subterminally at all sides of the axis. The neck is protruding and the egg chamber deeply sunken. One axis shows an archegonium and a bulging outgrowth with cells that are much larger than those of the axis (figure 5i). This has been interpreted as a gametophyte–sporophyte junction [15]. Another axis shows a similar outgrowth but no archegonium.

The organization of Langiophyton mackiei, the gametophyte of Horneophyton lignieri, is much more complex. Up to 1 cm long axes terminate in a 4–9 mm wide cup-shaped structure. Up to 30 vascularized projections arise from the bottom of the cup. The outer ones often are branched, whereas the inner are unbranched; some are finger shaped and others have a cup-shaped tip. These projections bear the archegonia. A projection may bear a single archegonium in the centre of the tip but in some branches archegonia are also positioned laterally. The archegonia have a massive neck and a deeply sunken egg chamber. The axis of antheridia-bearing gametophyte terminates in a bowl-shaped structure with numerous densely packed antheridia at the inner side of the bowl. Also Kidstonophyton discoides, the gametophyte of Nothia aphylla, of which only antheridia-bearing axes are known, is very complex (figure 5e). The axes show the typical surface structure of interrupted ridges formed by emergences and depressions as is seen in the aerial axes of Nothia. The axes end in an up to 8 mm wide disc-shaped gametangiophore with a slightly convex upper surface and curled up margins. A mixture of a large number of densely positioned sterile outgrowths and interspersed antheridia covers the upper surface of the gametangiophore. The antheridia are club- to flask-shaped. Very little is known about the developmental stages of Langiophyton and Kidstonophyton, except for some germinating spores of Horneophyton and very early cell divisions of the young gametophyte. The differences in morphology between the rather simple gametophytes of Aglaophyton and Rhynia on one hand and the very complex structure of the gametophytes of Horneophyton and Nothia on the other hand are striking. The former two species lived on the substrate, whereas the latter two had at least partly subterranean rhizomes.

For detailed descriptions of and discussions on gametophytes see:

Lyonophyton rhyniense: ([10–12,15,42]: germinating spores of Aglaophyton majus, [49,51,52]); Remyophyton delicatum: [15,49]; Langiophyton mackiei: ([13,15,44]: germinating spores, very young gametophytes, [52]); Kidstonophyton discoides: [15,52].

9. Asexual reproduction

Rhynie chert plants show several modes of asexual reproduction. Most common is clonal reproduction by repeatedly bifurcating rhizomes. All well-documented Rhynie chert plants provide evidence for clonal growth. In some cases, it is very obvious, e.g. in Nothia aphylla, which had subterranean rhizomes from which upright aerial axes departed [25,26]. Larger chert blocks show reasonably well-preserved as well as partly decayed upright aerial axes arising from the same rhizome system. The rhizomes that occur in several storeys over each other in the sandy soil are all well-preserved and show no signs of decay, some show some post-mortem shrinkage. They apparently belonged to the same plant. This is also an indication for certain seasonality of these perennial plants.

Another form of asexual reproduction is with propagules [18]. They developed from the epidermal cells surrounding the guard cells and from hemispherical projections of Rhynia gwynne-vaughanii. Isolated propagules may locally be common. Several developmental stages have been documented from small globose structures not showing a clear cellular differentiation to fully developed ovoid 337 µm–2.33 mm long and 418–499 µm wide vascularized propagules with stomata and rhizoids. Necrotic cells at the base are interpreted as an abscission layer and apical growth seems to begin after stomata have formed but prior to the formation of vascular tissue.

10. Concluding remarks

There are very few fossil plants, if any, known in such great detail as several of the Rhynie chert plants. However, not all taxa show an equal amount of detail. Some are very rare and comparatively poorly known. In some taxa only the basal parts are usually well preserved, whereas the soft tissues of the aerial axes are very poorly preserved or missing. This depends on the facies [53]. Nevertheless, even as a whole this flora is in many aspects most remarkable. Most of the Rhynie chert plants show a unique combination of characters unknown from any other land plant, including other Rhynie chert taxa. Phylogenetic analyses of Kenrick & Crane show that Aglaophyton and Horneophyton occupy a rather isolated position within the polysporangiophytes [54]. Some of the Rhynie chert plants show characters that are more typical for bryophytes than for vascular land plants, i.e. Horneophyton lignieri has columellate sporangia and uniformly thickened water-conducting cells, however, in the basal parts of the axes water-conducting cells have annular to helical thickenings. Horneophyton also shows other features that are not typically bryophyte-like but rather are found in vascular land plants such as well-developed cuticles with stomata on axes. Aglaophyton majus is considered to have a conducting system resembling leptoids and hydroids similar to those of some large modern mosses. However, phylogenetic analysis shows that this species is probably more closely related to vascular plants than to bryophytes [54]. New observations on the conducting system of Aglaophyton majus reveal that this species might indeed be closer related to Rhynia gwynne-vaughanii than currently thought. Nothia aphylla has laterally attached stalked sporangia that open into two valves like the zosterophylls, but the water-conducting cells lack thickenings. Two species, Trichopherophyton teuchansii and Ventarura lyonii, have laterally attached sporangia and true tracheids and are classified within the zosterophylls. The only plant that, from a modern perspective, would look more or less familiar is Asteroxylon mackiei. Its overall habit resembles that of the modern Huperzia selago, but Asteroxylon has stalked sporangia directly attached to the stem, whereas Huperzia has sporangia borne on sporophylls and vascularized true leaves.

Data accessibility

This article has no additional data.

Competing interests

I declare I have no competing interests.

Funding

The financial support of the German Research Foundation (DFG) is greatly acknowledged (Mo 412/13-1+2 and Ke 584/13-1 and 2).